Above-ground plant diseases are common in a wide variety of plants. These diseases are often caused by fungal or bacterial infections including infestations of Phytophthora infestans (late blight), Alternaria solani (early blight), Plasmopara viticola (downy mildew), Uncinula necator (powdery mildew), Puccinia recondita f.sp. tritici (leaf rust), Stagnospora nodorum (glume blotch), Pseudomonas syringae (bacterial speck), Botrytis cinerea (gray mold), Erwinia herbicola (bacterial blight) and Monilinia fructicola (brown rot). Phytophthora infestans (P. infestans) or "late blight" is best known as the fungus which caused the Irish potato famine. More than a century after it destroyed Ireland's potato crops, late blight is still a potentially devastating infestation affecting potato and tomato crops. The signs of a P. infestans infection include large, dark green, water-soaked spots on the plant's leaves and large, dark brown spots on the fruit or tuber. If left untreated, the leaves die and the potato or tomato rots.
Botrytis cinerea (B. cinerea), also known as gray-mold disease, is common on soft, ripe fruits (e.g., strawberries, raspberries); vegetables (e.g., lettuce, tomato, artichoke, asparagus); legumes (e.g., lima beans, kidney beans) and ornamental plants (e.g., african violet, begonia, carnation, rose, poinsettia). Signs associated with this fungal disease include a brown or gray dusty mold covering the fruit, stems or leaves.
To date, control of above-ground fungal or bacterial pathogens has been limited to treatment with synthetic chemical pesticides. Many chemical fungicides are classified as carcinogens by the EPA. Many of these chemicals are also toxic to wildlife and other non-target species. In addition, pathogens may often develop resistance to chemical pesticides. (See, e.g., Schwinn et al., p. 244, ADVANCES IN PLANT PATHOLOGY: PHYTOPHTHORA INFESTANS, THE CAUSE OF LATE BLIGHT OF POTATO (Academic Press, San Diego 1991)).
Biological control offers an attractive alternative or addition to present disease-control practices. It is safer for the environment and less expensive to produce. Biological control of above-ground diseases is, however, particularly challenging because the surface of a plant provides a difficult environment for the introduction, survival and multiplication of biological control agents such as bacteria. (See, e.g., Campbell, R., p.77, BIOLOGICAL CONTROL OF MICROBIAL PLANT PATHOGENS (Cambridge Univ. Press, New York 1989)). Thus, few commercially viable biocontrol agents are currently available for the control of above-ground plant disease. Bacillus subtilis is used commercially as a seed treatment (Kodiak.TM.), however, Bacillus sp. are not employed for above-ground plant diseases.
Naturally-occurring fungicidal bacterial strains are relatively uncommon. Screening programs have identified certain Bacillus sp. (B. sp. includes B. subtilis, B. cereus, B. mycodies, B. anthracis and B. thuringiensis) strains which exhibit antifungal activity. (See, e.g., Stabb et al. (1990) Applied Environ. Microbiol. 60(12):4404-4412). These strains have been shown to produce zwittermicin-A and/or "antibiotic B," now known as kanosamine (Milner et al. (1996) Appl. Environ. Microbiol. 62:3061-3065), two antibiotic agents which are effective against the soil borne disease "damping-off" caused by Phytophthora medicaginis, Phytophthora nicotianae, Phytophthora aphanidermatum or Sclerotinia minor. (See, Stabb et al., supra).
Zwittermicin A is a water-soluble, acid-stable linear aminopolyol molecule. (See, He et al. (1994) Tetra. Lett. 35(16):2499-2502). U.S. Pat. No. 5,049,379 to Handelsman et al. describes how zwittermicin A-producing B. cereus can be used to control the below-ground seedling diseases "root rot" and "damping off" in alfalfa and soybean. When the seed is coated with zwittermicin-A produced by B. cereus ATCC 53522, the pathogenic activity of the root rot fungus is inhibited. Similarly, application of spore-based formulations of certain B. cereus strains to soybean seeds or the soil surrounding the seeds has been shown to improve soybean yield at field sites. (See, Osburne et al. (1995) Am. Phytophathol. Soc. 79(6): 551-556).
Smith et al. (1993) Plant Disease 77(2):139-142 report that the activity of the soil-borne fungus, Pythium aphandiermatum, that causes cottony cucumber leak can be suppressed using zwittermicin-producing B. cereus strain UW85. Liefert et al. (1995) J. Appl. Bacteriol. 78:97-108 report the production of anti-Botrytis and anti-Alternaria brassicicola antibiotics by two Bacillus strains, B. subtilis CL27 and B. pumilis CL45. The whole broth and cell-free filtrates were active against Botrytis and Alternaria in in vitro tests and were active against Botrytis in in vivo small plants tests on Astilbe. The authors identified three antibiotics produced by the B. subtilis strain CL27, two of which were thought to be peptides and one non-peptide. Unlike the bacterial strains of the present invention, the antibiotics produced by the B. pumilis strain CL45 demonstrated only in vitro activity, no in vivo activity was demonstrated.
A lipoprotein antibiotic having a molecular weight of approximately 63,500 daltons that demonstrates activity against gram-positive bacteria but not gram-negative bacteria is disclosed in U.S. Pat. No. 5,061,495. In addition, this antibiotic is active against wheat powdery mildew caused by Erysiphe graminis and Botrytis fabae. McKeen et al. (1986) Phytopathol. 76:136-139 report the use of B. subtilis and antibiotics produced by B. subtilis for control of peach brown rot.
However, there have been no other reports of antibiotic-producing Bacillus sp. strains effective against pathogens responsible for above-ground plant diseases. Thus, there remains a need for biocontrol agents which are effective against above-ground fungal and bacterial infections.